Induced Fungal Resistance to Insect Grazing: Reciprocal Fitness Consequences and Fungal Gene Expression in the Drosophila-Aspergillus Model System

Abstract

BackgroundFungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and reproduction. Inducible resistance that is mounted after attack by fungivores may allow fungi to circumvent the potential costs of defence when not needed. However, no information exists on whether fungi display inducible resistance. We combined organism and fungal gene expression approaches to investigate whether fungivory induces resistance in fungi.Methodology/Principal FindingsHere we show that grazing by larval fruit flies, Drosophila melanogaster, induces resistance in the filamentous mould, Aspergillus nidulans, to subsequent feeding by larvae of the same insect. Larval grazing triggered the expression of various putative fungal resistance genes, including the secondary metabolite master regulator gene laeA. Compared to the severe pathological effects of wild type A. nidulans, which led to 100% insect mortality, larval feeding on a laeA loss-of-function mutant resulted in normal insect development. Whereas the wild type fungus recovered from larval grazing, larvae eradicated the chemically deficient mutant. In contrast, mutualistic dietary yeast, Saccharomyces cerevisiae, reached higher population densities when exposed to Drosophila larval feeding.Conclusions/SignificanceOur study presents novel evidence that insect grazing is capable of inducing resistance to further grazing in a filamentous fungus. This phenotypic shift in resistance to fungivory is accompanied by changes in the expression of genes involved in signal transduction, epigenetic regulation and secondary metabolite biosynthesis pathways. Depending on reciprocal insect-fungus fitness consequences, fungi may be selected for inducible resistance to maintain high fitness in fungivore-rich habitats. Induced fungal defence responses thus need to be included if we wish to have a complete conception of animal-fungus co-evolution, fungal gene regulation, and multitrophic interactions.

Phenotypic responses to microbial volatiles render a mold fungus more susceptible to insect damage

“…On the opposite side, mold conidia were point‐inoculated on banana medium; note that on this medium the A. nidulans wild‐type strain causes 100% larval mortality, and in the absence of beneficial microbes larvae do not develop at all, see Caballero Ortiz et al. () and Anagnostou et al. ().…”

“…The investigation of gene expression in A. nidulans had two purposes: (1) to explore the influence of yeast volatiles on key molecular regulators of morphological and chemical differentiation of A. nidulans ; and (2) to test whether volatile‐exposed mold loses its capacity to activate candidate “insecticidal defense” genes in response to insect damage (Caballero Ortiz et al., ). The expression of fungal secondary metabolite regulator genes was measured.…”

“…Purpose (i): A. nidulans colonies were prepared by spreading 100 μl conidia suspension on autoclaved cellophane foil placed on sucrose‐supplemented malt extract agar in 35‐mm plates. The foil allows diffusion of nutrients without affecting mold growth (Caballero Ortiz et al., ), but ensures sampling of pure fungal tissue without medium contaminations. The full fungal tissue was harvested with a scalpel.…”

“…Isolation of RNA from defined amounts of lyophilized A. nidulans tissue (100 mg per replicate), RNA quality checks, PCR conditions, and data analysis followed the protocol described in Caballero Ortiz et al. () (for more details and primers used see “Gene expression analysis”).…”

“…Experiments using mutant fungi showed that the mold's insecticidal properties are linked to their secondary metabolism (Trienens, Keller, & Rohlfs, ) (Figure ). The insecticidal properties are strong in strictly bipartite mold–larval interactions where other microbes were explicitly excluded (Trienens et al., ; Caballero Ortiz et al., ), whereas under more natural conditions that include the metabolic activity of yeasts, Drosophila larvae can hinder or even permanently suppress mold growth (Stamps et al., ; Rohlfs & Hoffmeister, ; Wertheim, Marchais, Vet, & Dicke, ). This latter observation prompted us to assume that in our tripartite model system insects are better able to keep mold in check because S. cerevisiae volatile metabolites interfere with the molecular and chemical mechanisms by which A. nidulans harms the insects in the absence of these volatiles.…”